ADP-ribosylation factors (ARFs) are about 20 kDa guanine nucleotide- binding proteins initially identified by their ability to enhance in vitro cholera toxin-catalyzed ADP-ribosylation and subsequently shown to participate in vesicular transport in the Golgi and other cellular compartments. ARFs are active in the GTP-bound form; hydrolysis of bound GTP to GDP, possibly with the assistance of a GTP hydrolysis (GTPase)-activating protein (GAP) results in inactivation. Exchange of GDP for GTP and reactivation were shown by other workers to be enhanced by Golgi membranes in a brefeldin A (BFA)-sensitive reaction, leading to the proposal that the guanine nucleotide-exchange protein (GDP) was a BFA target. In the studies reported here, a soluble GDP was partially purified from bovine brain. Exchange of nucleotide on ARFs 1 and 3, based on increased ARF activity in a toxin assay and stimulation of [35S]GTPgammaS binding was dependent on phospholipids, with phosphatidylserine being more effective than cardiolipin. GEP appeared to increase the rate of nucleotide exchange but did not affect the affinity of ARF for GTP. Whereas the crude GDP had a size of about 700 kDa, the partially purified GDP behaved on Ultrogel AcA 54 as a protein of 60 kDa. With purification, the GEP activity became BFA-insensitive, consistent with the conclusion that, in contrast to earlier inferences, the exchange protein is not itself the BFA target.